1. Field of the Invention
The present invention relates to transgenic sugar beet plants capable of tolerating herbicide treatment with glyphosate as active ingredient.
2. Background of the Related Art
Weeds in sugar beet fields are a major problem for the farmer. They compete with the crop thus reducing yield. Today, no single herbicide is able to effectively control all weeds without harming the sugar beet crop itself (Miller et al, J. Sugar Beets Res. 26: 3-4, 1989). In practice, farmers use mixtures of herbicides, which also reduce growth of the crop. Meanwhile the number of weed species having developed resistance to said herbicides continues to increase (Schweizer et al, J. of Sugar Beet Research 28: 1-23, 1991) thereby aggravating the problem of weed control in sugar beet fields.
Roundup(copyright) is a broadspectrum, environmentally preferable herbicide inhibiting the growth of both weed and crop species. In the context of the present invention one liter of a herbicidal Roundup(copyright) solution comprises 360 g of its active ingredient (a.i.) glyphosate (the common name of N-phosponomethyl-glycine) which is taken up by foliage. So far no glyphosate resistant weed has developed in over 20 years of use (Holt et al., Annu. Rev. Plant Physiol., 1993); additionally no natural tolerance to glyphosate has been found in sugar beet. However, pre-emergence use of Roundup(copyright) seems to be more efficient for weed control in sugar beet fields than a combination of herbicides often used in sugar beet agriculture, consisting of phenmediphan, metamitron and ethofumesate (Madsen et al, Weed Res. 35: 105-111, 1995).
Glyphosate inhibits the biosynthesis of aromatic amino acids, through irreversible binding to 5-enolpyruvylshikimate-3-phosphate synthase (epsps). Within the chloroplast this enzyme catalyzes the reaction of shikimate-3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate-3-phosphate and phosphate. Approximately one week after application of the herbicide, visible effects can be seen including wilting, yellowing followed by complete browning, deterioration of plant tissue, and decomposition of the roots.
To impart glyphosate tolerance to crop species, focus has been on the introduction into plants of epsps genes capable of increasing glyphosate tolerance. Besides plants bacteria and fungi naturally express epsps enzyme activity. The cp4/epsps from Agrobacterium sp. CP4 was found to confer tolerance to glyphosate (Barry et al., xe2x80x9cInhibitors of Amino Acid Biosynthesis: Strategies for Imparting Glyphosate Tolerance to Crop Plantsxe2x80x9d, in: Biosynthesis and Molecular Regulation of Amino Acids in Plants, Singh et al (eds), American Society of Plant Physiologists, pages 139-145, 1992). Introduction of the cp4/epsps gene into soybean and oilseed rape yielded tolerance to foliar application of the herbicide under field conditions (Delannay et al., Crop Sci. 35: 1461-1467, 1995; Padgette et al., Crop Sci. 35: 1451-1461, 1995).
Glyphosate oxidase reductase (gox) isolated from Achromobacter sp. strain LBAA (Barry et al., supra) degrades glyphosate into aminomethyl phosphonic acid, a compound non-toxic for the plant. A combination of the cp4/epsps and glyphosate oxidase (gox) genes has been successfully used to obtain transgenic wheat (Zhou et al., Plant Cell Rep. 15: 159-163, 1995) tolerant to glyphosate.
The object of the present invention is to provide sugar beet plants which tolerate glyphosate in doses sufficiently high to effect optimal herbicidal activity. Such plants can be further improved by backcrossing with elite sugar beet lines to optimize agronomic properties such as yield, pathogen resistance, etc.
Sugar beets may be transformed using Agrobacterium tumefaciens mediated transformation (Fry et al, Third international congress of plant mol. biol., Tuscon, Ariz., USA; D""Halluin et al, Bio/Technology 10: 309-314, 1992; Konwar, J. Plant Biochem and Biotech 3: 37-41,1994). Agrobacterium-mediated transformation often results in more than one copy of the T-DNA being integrated into the plant""s genome. The gene to be integrated is preferably introduced into the T-DNA such that it becomes located close to the T-DNA right border which, contrary to the left border, will almost always be transferred to the plant.
Plants according to the present invention tolerate treatment with more than about 3xc3x976 liters of the herbicide Roundup(copyright) per hectar (about 18 liters per hectar). The total standard dose to obtain good weed control varies between 4 and 6 liters per hectare, depending on weed pressure. At these concentrations herbicide treatment exerts no detectable effect on plant vigour and leaf chlorosis. The tolerance exhibited by the plants according to the invention is conferred by a transgenically expressed cp4/epsps enzyme activity. A preferred embodiment of the present invention has been deposited with the National Collections of Industrial and Marine Bacteria Limited (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland UK, on Oct. 24, 1997, under the Accession No. 40905. This deposit will be maintained in the NCIMB depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request or for the effective life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant has satisfied all the requirements of 37 C.F.R xc2xa7xc2xa71.801-1.809, including providing an indication of the viability of the sample. Applicant imposes no restriction on the availability of the deposited material from the NCIMB; however, Applicant has no authority to waive any restriction imposed by law on the transfer of biological material or its transportation in commerce.
The present invention thus relates to a sugar beet plant including the descendants thereof expressing cp4/epsps enzyme activity. In particular the invention relates to a sugar beet plant including the descendants thereof tolerating the treatment with about 4 to about 18 liters Roundup(copyright) per hectar.
Plants according to the present invention can be obtained by routine Agrobacterium mediated transformation using a transformation vector comprising between right and left T-DNA border sequences a piece of DNA as described in SEQ ID NO: 5 encoding i.a. cp4/epsps.
It was surprisingly found within the scope of the present invention, that a transformation event (RRMax) lacking left and right T-DNA border sequences within the transgenic genome and resulting in deletion of a considerable part of the transformation vector DNA while retaining the cp4/epsps encoding DNA provides superior glyphosate tolerance. In particular a piece of DNA as characterized by SEQ ID NO: 1 is found integrated into a highly repetitive region of the genome simlutaneously replacing part of said repetitive genomic sequence. The genomic DNA directly adjacent to that part of the transgene sequence which in the transformation vector used is linked to the T-DNA right border sequence, has the sequence given in SEQ ID NO: 2. The genomic DNA directly adjacent to the other end of the integrated transgenic DNA has the sequence given in SEQ ID NO: 3. The complete DNA sequence of the newly formed genomic DNA arrangement is given in SEQ ID NO: 4.
Accordingly, the present invention relates to a sugar beet plant including the descendents thereof wherein DNA characterized by the nucleotide sequence of SEQ ID NO: 1 forms part of the plant""s genome and said nucleotide sequence preferably replaces highly repetitive DNA sequences within the plant""s genome.
Preferred herein is a sugar beet plant including the descendents thereof wherein those parts of the genome directly linked to said nulceotide sequence are characterized by the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
The herbicide tolerance engineered into the transgenic seeds and plants mentioned above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in descendant plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. As the growing crop is vulnerable to attack and damages caused by insects or infections, measures are undertaken to control plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of infected plants, as well as the application of agrochemicals such as fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.
Use of the herbicide tolerance of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate descendant plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained which, due to their optimized genetic xe2x80x9cequipmentxe2x80x9d, yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.
In seeds production germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD(copyright)), methalaxyl (Apron(copyright)), and pirimiphos-methyl (Actellic(copyright)). If desired these compounds are formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.
It is a further aspect of the present invention to provide new agricultural methods such as the methods exemplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.
In another embodiment, the present invention relates to a transgenic plant cell, tissue, organ, seed or plant part obtained from the transgenic plant. Also included within the invention are transgenic descendants of the plant as well as transgenic plant cells, tissues, organs, seeds and plant parts obtained from the descendants.
The invention further relates to a commercial bag containing Roundup(copyright) tolerant sugar beet seed capable of expressing cp4/epsps, together with lable instructions for the use thereof. Preferred within this invention is a commercial bag comprising seed of a transgenic plant comprising stably integrated into its genome a piece of DNA having the nucleotide sequence depicted in SEQ ID NO: 1.
The transformation methodology employed can be summarized as follows:
(a) transforming in vitro grown sugar beet cotyledons using Agrobacterium tumefaciens with a vector comprising a piece of DNA encoding cp4/epsps such as described in SEQ ID NO: 5;
(b) regenerating shoots in the presence of glyphosate;
(c) transferring the shoots to soil in the greenhouse;
(d) treating plantlets with glyphosate;
(e) visually grading plant vigour and leaf chlorosis;
(g) selecting completely normal plants with normal vigour and leafes uneffected by glyphosate treatment; and
(h) propagating the selected plants using conventional breeding techniques.
In particular shoots are regenerated in the presence of about 0.1 to about 10 mM, preferably about 1 mM glyphosate after 8-12 weeks of selection. Each transgenic shoot is further propagated into ten copies which can optionally be analyzed for the presence of the cp4/epsps transgene using polymerase chain reaction (PCR) before it is transferred to the greenhous for rooting. Light conditions in the greenhouse are 16 hours of light and 8 hours of darkness with a temperature of 22xc2x12xc2x0 C. At the three to four leaf stage, the plantlets are sprayed with an aqueous solution of Roundup(copyright) at a dose of 0.1 to 20 liters, preferably 1 liter per hectar. Visual injury ratings for plant vigour and plant chlorosis based on a scale from 0 (dead plant) to 9 (completely uneffected plant) are taken on individual plants 2 weeks after glyphosate application. Ratings of 0 to 3 are characteristic of susceptible plants. Ratings of 3 to 7 indicate a low to intermediate level of tolerance, and ratings of 8 or 9 indicate good levels of tolerance. In particular the the ratings have the following meaning:
To collect data from field trials plants with normal vigour and uneffected by glyphosate treatment (rating 9) are further propagated or bred by convential techniques.
Transformation is preferably performed using a Ti vector comprising a piece of DNA with the sequence given in SEQ ID NO: 5 containing the cp4/epsps and gox genes both of which have been reported to confer tolerance to glyphosate in certain plant species, and the reporter gene uidA encoding the xcex2-glucuronidase enzyme. The enhanced 35S promoter (Odell et al., Nature 313: 810-812, 1985) is linked to the uidA gene, the figworth mosaic virus (FMV) promoter (Gouwda et al., J. Cell. Biochem. Suppl. 13D: 301, 1989) to the cp4/epsps and gox genes. Upstream of the cp4/epsps and the gox genes a transit peptide (Gasser et al., J. Biol. Chem. 263:4280-4289, 1988) is inserted to achieve targeting of both of the proteins to the chloroplast.
One transformation event which was generated in accordance with the present invention is surprisingly found to be uneffected by doses of up to about 3xc3x976 liters (about 18 liters) of the herbicide Roundup(copyright) per hectar. Molecular analysis reveals that
there is a single copy of transgenic DNA integrated at a single locus;
the integrated DNA encodes cp4/epsps and corresponds to truncated vector DNA;
the integrated DNA replaces a piece of genomic DNA and has the sequence shown in SEQ ID NO: 1;
the genomic DNA directly adjacent to the integrated DNA is characterized by the sequences of SEQ ID NO: 2 and SEQ ID NO: 3 resulting in the new genomic sequence arrangment of SEQ ID NO: 4;
the cp4/epsps and the uidA genes are intact whereas the gox gene is truncated;
other vector sequences are not present in the transgenic plants.
Now that this information is available plants derived from one of the specific transformation events according to the invention can be easily distinguished from other sugar beet plants by means of PCR. Suitable primer pair combinations allow to specifically identify genomic DNA sequences which are only present in plants directly or indirectly resulting from identical transformation events. Such events are not in any way limited to those obtained by Agrobacterium-mediated transformation but may also result from biolistic transformation experiments.
The present invention thus further relates to a sugar beet plant including the descendents thereof characterized in that PCR amplification with its genomic DNA as template results in amplification of
a 739 bp DNA fragment when using a pair of oligonucleotide primers characterized by the sequences of SEQ ID NO: 18 and SEQ ID NO: 21; or
a 834 bp DNA fragment when using a pair of oligonucleotide primers characterized by the sequences of SEQ ID NO: 20 and SEQ ID NO: 24; or
a 1057 bp DNA fragment when using a pair of oligonucleotide primers characterized by the sequences of SEQ ID NO: 17 and SEQ ID NO: 22; or
a 1224 bp DNA fragment when using a pair of oligonucleotide primers characterized by the sequences of SEQ ID NO: 19 and SEQ ID NO: 25.